With the information gained by the sequencing of the human genome,1,2 gene therapy now holds promise for the treatment of hereditary diseases and cancers.3,4 It is now possible to silence a bad gene5, turn on a needed gene6, or install a new gene to address particular cellular defects.7 However, a key requirement for successful gene therapy is the efficient transfer of DNA to specific cell types in vivo. Viral vectors have proven to be very efficient transfection agents and allow for the insertion of foreign DNA into many cell types.8,9 However, the inherent problems with viral vectors such as immunogenicity and the limited size of the DNA plasmid that can be transferred has led to interest in developing efficient non-viral vectors.10-14 Non-viral vectors are ideal because of their expected low toxicity and immunogenicity, ability to transfer large strands of DNA and simpler synthetic preparation. Typically, these vectors consist of a lipophilic component attached to a positively charged, polar headgroup, through the use of a spacer or linking motif, and are a mixture of neutral and positively charged lipids. A wide range of cationic liposomes have been synthesized and recently reviewed.15-17 The cationic headgroups typically found in these liposome systems include quaternary ammonium salts,10 polylysine,18 polyguanadinium salts,19 polyarginine20 and polyamines.21 
Polyamines were introduced into liposomes by Behr,22 when it was realized that the naturally-occurring polyamines such as spermidine and spermine (FIG. 1) could efficiently compact DNA. Behr went on to synthesize one of the first transfection vectors, DOGS 1, and incorporated a branched polyamine as the polar headgroup (FIG. 2).11 
Following this work, a number of research groups have looked at the effect of changing the positively-charged, polyamine headgroup on cationic liposomes with respect to their efficiency at transfecting DNA.15,16,21,23,24 Byk et al21 showed that when assayed in HeLa cells, the compound RPR 120535 2, whose configuration of the polar head is linear, was 5-10 times more effective at transfection than those with branched-, globular- or T-shaped polar domains. Safinya et al24 demonstrated with 3 that lipids containing a higher number of positive charges had better transfection efficiency within a series of liposomes containing branched-polyamine headgroups. Other popular vectors 4-6 are shown in FIG. 2. Commercially-available Lipofectamine is a 3:1 mixture of 4 and 6.18b 
Although a large number of cationic lipids have been surveyed, a systematic study of how linear polyamine architectures effect transfection efficiency is still needed, especially in light of the molecular recognition elements required to use the polyamine transporter (PAT) for cellular entry.25-32 Our goal was to perform the key crossover experiments needed to tie these two fields together. Indeed, understanding how cationic motifs are transported across the cell membrane is critical to both enterprises.
Targeting a specific cellular transporter could provide cell-selective transfection. For example, rapidly-proliferating cancer cells could be targeted via their PAT, which is often up-regulated.28 Indeed, the ability to transfect a specific cell type would have profound impact on a multitude of gene therapy strategies.
Since many cancer cell lines have active polyamine transporters, it is possible to target these cells using the molecular recognition events involved in polyamine import.28 For example, an anthracene-homospermidine conjugate was shown to be 10-30 fold more toxic to B16 melanoma cells than to ‘normal’ melanocytes (Mel-A cells).28 A multitude of polyamine structures were previously screened for their high PAT selectivity in CHO and CHO-MG cells.28 Several linear polyamine architectures were identified, which selectively targeted the PAT-active CHO cell line over its PAT-inactive CHO-MG mutant.25-32 The discovery of homospermidine, a 4,4-triamine, as a cell-selective ‘vector’ motif provided the means to test the PAT-delivery system as a conduit for gene delivery.26,28 
In short, our aim was to combine these two areas of research (PAT targeting and gene delivery) by attaching PAT-targeting polyamine sequences28 to aryl lipid motif 324 in order to facilitate DNA plasmid uptake. These materials were then evaluated for their DNA-transporting ability as well as transfection efficacy and compared to the commercially-available transfection reagent, Lipofectamine 2000.33 